Method of improving chloroplast function and increasing seed yield

ABSTRACT

Ascorbate protects tissues against damage caused by reactive oxygen species (ROS) produced through normal metabolism or generated from stress. The inositol route to AsA involves four enzymes: myo-inositol oxygenase, glucuronate reductase, gluconolactonase (GNL), and  L -gulono-1,4-lactone oxidase (GulLO). Eighteen putative GNLs were identified in  Arabidopsis , one of which, AtGNL, is interesting because it possesses a chloroplastic signal peptide. Knockouts on this gene had lower foliar AsA and stunted growth compared to controls. The functional gene restored the phenotype of the knockouts, and those plants had higher AsA content, enhanced photosynthetic capacity, and higher seed yield.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation in part ofU.S. Patent Application No. 62/428,775 entitled “Method of ImprovingChloroplast Function” filed on Dec. 1, 2016.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-WEB as an ASCII (.txt) formatted sequence listing with a filenamed 2018-01-11-US_ST25.txt, created on Jan. 11, 2018 and having a sizeof 14 KB and accompanies this specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety and includes nonew matter.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO A MICROFICHE APPENDIX

Not Applicable.

RESERVATION OF RIGHTS

A portion of the disclosure of this patent document contains materialwhich is subject to intellectual property rights such as but not limitedto copyright, trademark, and/or trade dress protection. The owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent files or records but otherwise reserves all rightswhatsoever.

BACKGROUND OF THE INVENTION 1. Field of the Invention

1.1. Vitamin C

Vitamin C (a.k.a. L-ascorbic acid, AsA) is the most abundantwater-soluble antioxidant found in plants (Smirnoff, 2000). In the late1750's it was established that citrus fruits contained a “cure” forscurvy a.k.a. “sea plague”. However it was 1928 when Albert Szent-Gyôryiisolated ascorbate and identified it as the anti-scorbutic componentpresent in citrus and other fruits. Since then, a significant body ofevidence has been accumulated about the importance of vitamin C forhuman and animal health (Drouin et al., 2011; Padayatty, 2016).

Ascorbate is a non-enzymatic antioxidant with a simple molecularstructure. As is true for many other antioxidants, vitamin C is presentin plants in two forms, the reduced and most active form calledascorbate and the oxidized form named dehydroascorbate. The chemicalstructure of ascorbate (AsA) and dehydroascorbate (DHA), the reduced andoxidized forms of vitamin C, are shown in FIG. 1. Ascorbate belongs tothe family of six carbon sugars with a conjugated pi enediol system atcarbons 2 and 3. Ascorbate is a very effective antioxidant due to itsability to donate a pair of electrons and stabilize the subsequentcharge using the two oxygen atoms at C2 and C3 in the oxidized form ofthe molecule (Lee et al., 2006). Ascorbate eliminates reactive oxygenspecies (ROS) from both inside and outside cells, because it is able topass through the cell membrane in its oxidized form where it reduces thelipophilic α-tocopherol (vitamin E) which is membrane bound in itsoxidized state. This makes ascorbate an important part of the enzymaticantioxidant cycle in recycling other important antioxidants which wouldotherwise be lost to the cell (Traber and Stevens, 2011).

1.2. Importance of Ascorbate in Humans

Humans, primates, and a few other animals including teleost fishes,guinea pigs, some bats and passeriforme birds cannot synthesize vitaminC due to of the lack of an active L-gulono-1,4-lactone oxidase (GuILO)(Drouin et al., 2011). This enzyme catalyzes the last step in vitamin Cbiosynthesis, and it is highly mutated and non-functional in thesespecies (Sato et al., 1976). Humans and other animals need to consumethis essential vitamin from fresh fruits and vegetables to satisfy theirneeds.

In animals, vitamin C is involved in the synthesis of collagen, animportant component of the skin, scar tissue, tendons, ligaments, andblood vessels (Levine et al., 1995; Davey et al., 2000). Anotheressential role of vitamin C is related to its function in redoxhomeostasis; this means when the production of ROS increases, the body'sresponse will increase the activity of the endogenous antioxidant systemthrough redox signaling (Figueroa-Méndez and Rivas Arancibia, 2015).Vitamin C functions in oxidative protein folding and in the maintenanceof the intraluminal oxidative environment, which suggests that it has aparticular role in endoplasmic reticulum related processes (Mandl etal., 2009). In animals, vitamin C interacts enzymatically andnon-enzymatically with ROS. In humans vitamin C is essential inpreventing pathological conditions including cardiovascular disease,cancer, hepatitis, bacterial infections, fungal infection, and allergies(Cathcart, 1981; Padayatty et al., 2006). The two-time NobelPrize-winner, Linus Pauling, demonstrated that cancer patients treatedwith high doses of vitamin C had an increased survival rate (Cameron andPauling, 1976). Recently Yun et al., (2015) showed that high levels ofvitamin C killed human colorectal cancer cells. This effect is due toincreased uptake of dehydroascorbate, the oxidized form of the molecule.Vitamin C has a uricosuric effect in humans and decreases uric acidlevels, exerting a protective effect on gout (Stamp et al., 2013).

1.3. Importance of Ascorbate in Plants

In plants, ascorbate (AsA) has a wide variety of physiological roles. Itfunctions as an enzyme cofactor, as a radical scavenger, and asdonor/acceptor of electron transport in the chloroplast (Conklin andBarth, 2004; Ishikawa et al., 2006). Ascorbate can protect tissuesagainst damage caused by ROS produced through normal oxygenic metabolismor generated from biotic and abiotic stress, and is strongly associatedwith photosynthesis and respiration. Reactive oxygen species includemolecules such as superoxide and hydrogen peroxide.

Chloroplasts as well as mitochondria produce ROS as byproducts of normalcellular metabolism, but this production is enhanced by a variety ofenvironmental stresses (Conklin et al., 1996; Conklin and Barth, 2004).Another essential role of AsA is the modulation of processes such aslignification, cell division, cell elongation, the hypersensitiveresponse, tolerance to stresses, and senescence in plants (Smirnoff andWheeler, 2000; Barth et al., 2004; Pavet et al., 2005). In addition, AsAcontrols flowering time through phytohormones (Barth et al., 2004).Ascorbate can accumulate at millimolar concentrations in bothphotosynthetic and non-photosynthetic tissues (Foyer et al., 1983). Thisaccumulation in such high quantities suggests that AsA is important forthe plant as a major antioxidant.

1.4. The Ascorbate Metabolic Network

The biosynthetic pathway for vitamin C in animals was elucidated in theearly 1950s and was proposed based on in vivo radio-labelling andfeeding experiments in rats (Ishikawa et al., 2006). There is a singlebiosynthetic pathway for vitamin C in animals. Evidence obtained duringthe last 18 years indicates that there are four pathways that lead tothe formation of AsA in plants. These routes are theD-mannose/L-galactose (Wheeler et al., 1998), L-gulose (Wolucka and VanMontagu 2003), D-galacturonate (Agius et al., 2003), and myo-inositol(Lorence et al., 2004) pathways. FIG. 2 shows this ascorbate metabolicnetwork.

1.4.1. The D-Mannose/L-Galactose Pathway

It is commonly known as the “Smirnoff/Wheeler” pathway. All the genesinvolved in this route have been characterized. The starting precursorfor this route is D-glucose, which is converted to GDP-D-mannosefollowed by L-galactose that leads to AsA production. D-Glucose-6-P isthen converted to GDP-D-mannose by a series of steps catalyzed byfructose-6-P, D-mannose-6-P, and D-mannose-1-P, and that include thevtc1 mutant locus (GDP-mannose pyrophosphorylase). All of these enzymeshave been cloned and characterized (Conklin et al., 1999; Qian et al.,2007; Maruta et al., 2008). The conversion of GDP-D-mannose to AsAcomprises four steps: GDP-L-galactose, L-galactose-1-P (Laing et al.,2004), L-galactose (Gatzek et al., 2002) and L-galactono-1,4-lactone(Imai et al., 1998). The GDP-D-mannose to GDP-L-galactose reaction iscatalyzed by GDP-D-mannose-3′,5′-epimerase a.k.a. GME (Wolucka and VanMontagu, 2003). The vtc2 enzyme and its close homolog vtc5 convertGDP-L-galactose into L-galactose-1-P (Smirnoff et al., 2001; Dowdle etal., 2007; Linster et al., 2007). The L-galactose-1-phosphatephosphatase (locus vtc4 in Arabidopsis) converts L-galactose-1-P intoL-galactose (Conklin et al., 2006). L-Galactose is oxidized at theC1-position by an L-galactose dehydrogenase (GaIDH) present in thecytosol to L-galactono-1,4-lactone (Wheeler et al., 1998; Gatzek et al.,2002). All enzymes in this pathway are cytosolic except the last stepinvolving the oxidation of galactono-1-4-lactone to AsA that is carriedout by the mitochondrial L-galactono-1,4-lactone dehydrogenase (GLDH),(Østergaard et al., 1997; Imai et al., 1998).

1.4.2. The L-Gulose Pathway

The L-gulose pathway uses a similar precursor as theD-mannose/L-galactose to the branch point at GDP-D-mannose.GDP-D-mannose is then converted by the GME enzyme into GDP-L-gulose. Itis proposed that GDP-L-gulose is converted to L-gulono-1,4-lactone andto AsA in three subsequent steps catalyzed by GDP-L-gulosepyrophosphatase, L-gulose-1-phosphate phosphatase, and L-gulosedehydrogenase respectively (Wolucka and Van Montagu, 2003). The onlyenzyme that has been characterized in this pathway is GME and it isknown to be cytosolic.

1.4.3. The D-Galacturonic Acid Pathway

Observations made in ripening strawberries established thatD-galacturonic acid and its methyl ester can be metabolized to form AsA(Mapson and Isherwood, 1956, Loewus and Kelly, 1961). In addition tothese early observations the discovery of a D-galacturonic acidreductase (GaIUR) gene from strawberry (Agius et al., 2003) provide themain evidence supporting this route. To date GaIUR is the only enzymeidentified in this route, which has been shown to be cytosolic (Agius etal., 2003).

1.4.4. The Myo-Inositol Pathway

Biochemical and molecular data indicate that myo-inositol can also be aprecursor for the biosynthesis of AsA in Arabidopsis (Lorence et al.,2004). This pathway involves four enzymes, starting from the oxidationof myo-inositol to D-glucuronic acid and further reduction to L-gulonicacid and to L-gulono-1,4-lactone, and further oxidation to AsA. Theseconversions are catalyzed by myo-inositol oxygenase (MIOX), glucuronatereductase (GlcUR), gluconolactonase (GNL), and L-gulono-1,4-lactoneoxidase (GuILO) respectively. The first two enzymes have alreadycharacterized by the Lorence Laboratory. Arabidopsis thaliana linesover-expressing MIOX and GulLO are tolerant to multiple abiotic stressessuch as salt, cold, heat, and pyrene (Lisko et al., 2013). These highexpressing AsA plants exhibit increased growth and biomass accumulation(Lorence et al., 2004; Lorence and Nessler, 2007; Lisko et al., 2013).

Transgenic rice overexpressing MIOX showed improved growth performancewhen grown in the presence of 200 mM mannitol and presented highersurvival rates compared to wild type plants treated with polyethyleneglycol (Duan et al., 2012). MIOX proteins are present in almost allmulticellular eukaryotes and are highly conserved across phyla. It hasbeen reported that the role of MIOX and D-GlcUA for AsA biosynthesis inplants is a major plant antioxidant to counterbalance oxidative damage(Shao et al., 2008). The first two enzymes in the inositol pathway toAsA are cytosolic. The Lorence laboratory has evidence indicating thatsome isoforms of the last two enzymes in this pathway reside in thechloroplast and the endoplasmic reticulum (ER).

1.5. Role of Ascorbate in the Chloroplast

Chloroplasts are the organelles responsible for photosynthesis, aprocess that is essential for plant growth and development (Rustchow etal., 2008; Venkatasalam, 2012). Key metabolites in the photosyntheticprocess are NADPH and ATP. Although photosynthesis is an essentialprocess, light absorption creates oxidative stress due to the formationROS, such as singlet oxygen (¹O₂), superoxide (O₂ ⁻) and hydrogenperoxide (H₂O₂) (Oelze et al., 2008). Under high light, the electronflow through the photosynthetic chain overcomes the passage of electronsfrom ferredoxin to several reductases, and this causes an over-reductionof the plastoquinone and cytochrome b complex. Thus, during a day withhigh irradiance, plants are under constant oxidative stress (Oelze etal., 2008). Light/dark cycles are probably the most important signalsthat regulate plant development. Light is essential for photosynthesis,but an excess inside the chloroplast leads to excessive ROS. Among thechief defense mechanisms that allow plants to cope with environmentalstress situations is the ascorbate-glutathione cycle, a complexmetabolic pathway in which a variety of photochemical and enzymaticsteps are involved. Ascorbate is essential to detoxify H₂O₂ producedduring the Mehler reaction, which is formed by dismutation of O₂ ⁻ andcan be regenerated via the AsA-glutathione cycle to counteract O₂ ⁻(Halliwell and Foyer, 1976; Foyer and Noctor, 2000; Munné-Bosch andAlegre, 2002; Talla et al., 2011). FIG. 3 shows the Anti-oxidation ofreactive oxygen species. Reactive oxygen is generated when electrons(e−) not utilized in photosynthesis are donated to oxygen, thus creatingsuperoxide (O2.−) that can be converted to hydrogen peroxide (H2O2) bysuperoxide dismutase (SOD). The H2O2 is further converted to H2O byascorbate peroxidase (APX) utilizing ascorbate (AsA) as an electrondonor that, in turn, becomes oxidized ascorbate (ox-AsA). Additionalelectrons are consumed via the conversion of ox-AsA back to AsA or theconversion of double ox-AsA back to AsA using glutathione. The resultingoxidized glutathione is reduced by electrons from electron transport bymeans of glutathione reductase (GR). Source: Demmig-Adams et al., 2012.The demand for AsA in these reactions increases at higher lightintensities, when formation of ROS is enhanced. Experimental evidencesuggests the existence of an effective signaling network between thechloroplast and the mitochondria that involves ROS and antioxidants(Foyer and Noctor, 2003; Noctor et al., 2007). The different light/darkconditions are currently one of the major challenges in plant researchto improve crop productivity under a changing global climate.

Ascorbate is present in all plants although its concentration variesgreatly and has been identified in various compartments of the cell.Ascorbate occurs inside as well as outside the chloroplast (Constable,1963; Hall and Rao, 1999, Habermann, 2013), where it has been shown toaccumulate at concentrations up to 50 mM (Hall and Rao, 1999); thisrepresents about 25-30% of the total AsA in the plant cell (Horemans etal., 2000). All known AsA biosynthetic enzymes reside in compartmentsother than the chloroplast, and therefore it is currently unknown howthis organelle is able to accumulate such high concentrations of thisantioxidant.

Ascorbate was at one time considered to be a necessary component of thephotosynthetic phosphorylation system (Arnon, 1959) however is nowconsidered important in providing a protective role in preventinginactivation of essential components of the chloroplasts (Pintó-Marijuanand Munné-Bosch, 2014).

It has been recognized for more than a century that chloroplasts altertheir distribution within cells depending on the external lightconditions. Chloroplasts can be observed to move to positions thatmaximize photon absorption under low-influence light and, conversely, tomove to positions that minimize photon absorption under high light. Themovement away from areas of strong light is believed to offer areas ofstrong light protection against photo-oxidative damage (Eckardt, 2003).

Arabidopsis plants growing under long day conditions (12 h ofphotoperiod) accelerate flowering in comparison with plants growingunder shorter photoperiods. Short days distinctly extend the vegetativephase of Arabidopsis growth and delay senescence (Lepistö and Rintamäki,2012). In the course of high-light acclimation, elevated ROS productionis compensated for by induction of antioxidant systems in leaves whichin turn prevent the oxidation of leaf cells (Mittler et al., 2004).

1.6. Definition of the Problem

Significant progress has been made in the characterization ofmyo-inositol oxygenase (MIOX) and glucuronate reductase (GlcUR), thefirst two enzymes of the myo-inositol pathway to AsA (Lorence et al.,2004; Lorence and Nessler, 2007). High AsA lines over-expressing MIOX4and GuILO are tolerant to multiple causes of oxidative stress includingsalt, cold, heat, and pollutants (Lisko et al., 2013). The third enzyme,GNL, has been characterized in rat, Zymomonas mobilis, and Pseudomonasaeruginosa (Tarighi et al., 2008), but not in plants. Current researchin the Lorence Group focuses on the characterization of this thirdenzyme. Preliminary data indicate the presence of isoforms ofglucuronolactonase (GNL) that are targeted to the ER and thechloroplasts. This project focuses on gaining insights about thefunction of a putative GNL that possesses a chloroplastic signalpeptide. This is quite relevant as it is currently unknown how thisorganelle that makes photosynthesis possible, is able to accumulate upto 50 mM AsA.

1.7. Hypothesis and Aims for the Role of Ascorbate in the Chloroplast

If At1g56500 encodes a functional glucuronolactonase (GNL) that residesin the chloroplast, then this protein will protect this organelle andgreen tissues, and will counteract reactive oxygen species formed underlight stress. When over-expressed in plants this enzyme will conferplants enhanced photosynthetic efficiency.

This hypothesis will be tested by addressing the following aims:

Aim 1: Characterize the AtGNL (At1g56500) functional enzyme.

Aim 2: Establish the role of the AtGNL under low, normal, and high lightconditions.

Aim 3: Characterize the phenotype and photosynthetic efficiency ofArabidopsis lines with low, normal, and high AtGNL expression.

II. Description of the Known Art

Patents, patent applications, and references disclosing relevantinformation are disclosed below. These patents, patent applications, andreferences are hereby expressly incorporated by reference in theirentirety.

SUMMARY OF THE INVENTION

Chloroplasts, the organelles responsible for photosynthesis, areessential for plant growth and development, and are involved in themetabolism of carbon, nitrogen, and sulfur (Venkatasalam, 2012; Rustchowet al., 2008). In addition, chloroplasts synthesize amino acids, fattyacids, purine, and pyrimidine bases, isoprenoids, tetrapyrroles, and thelipid components of their own membranes, followed by processing,folding, and assembly by various chaperone systems (Peltier et al.,2006). The chloroplasts need considerable protein import from thecytosol. Chloroplasts control nuclear gene expression indirectly bymetabolites, ROS and other cellular processes (Pogson et al., 2008;Pfannschmidtm, 2010).

It has been recognized for more than a century that chloroplasts altertheir distribution within cells depending on the external lightconditions. Chloroplasts can be observed to move to positions thatmaximize photon absorption under low light and, conversely, to move topositions that minimize photon absorption under high light. The movementaway from areas of strong light is believed to offer protection againstphoto-oxidative damage (Eckardt, 2003). The photoreceptors responsiblefor light induced chloroplast movement in higher plants arephototropins. The phototropins PHOT1 and PHOT2 are involved in bluelight mediated chloroplast relocation, stomatal opening and phototropism(Briggs and Christie, 2002). PHOT1 is the primary photoreceptor thatcontrols phototropism in low light (Huala et al., 1997), whereas PHOT2is responsible for the light-avoidance relocation of chloroplast underhigh light (Kagawa et al., 2001).

Ascorbate (AsA) is found in all plants although its concentrations varygreatly. Within the leaf, ascorbate occurs inside as well as outside thechloroplasts (Constable, 1963; Habermann, 2013). Ascorbate was at onetime considered to be a necessary component of the photosyntheticphosphorylation system but recently it has been regarded as having aprotective role in preventing inactivation of essential components ofthe chloroplasts (Arnon, 1959; Pintó-Marijuan and Munné-Bosch, 2014).Ascorbate helps detoxify H₂O₂ produced during the Mehler reaction,(Foyer and Noctor 2000; Talla et al., 2011) and is important forphotoprotection (Demmig-Adams et al., 2012). In chloroplasts, highascorbate levels are required to overcome photoinhibition caused bystrong light (Miyaji et al., 2014).

The myo-inositol pathway is one of the four routes for the production ofAsA in plants. This pathway has not been completely elucidated. Threeenzymes have been characterized: myo-inositol oxygenase (MIOX),glucuronate reductase (GlcUR), and L-gulono-1,4-lactose oxidase (GuTLO)(Lorence et al., 2004; Lorence and Nessler, 2007, Lisko et al., 2013;Aboobucker, 2014). The third enzyme, gluconolactonase (GNL), has beencharacterized in Rattus norvegicus (Kondo et al., 2006), Euglenagracilis (Ishikawa et al., 2008), Pseudomonas aeruginosa (Tarighi etal., 2008), Xanthomonas campestri (Chen et al., 2008), Homo sapiens(Aizawa et al., 2013), and Gluconobacter oxydans (Shinagawa et al.,2009), but not in plants.

The first two enzymes in this pathway (MIOX4 and GlcUR) are cytosolic,the fourth enzyme (GuILO) resides in the ER as illustrated in FIG. 4.FIG. 4 shows the Subcellular localization of enzymes in the ascorbatemetabolic network. GLDH, the terminal enzyme in theD-mannose/L-galactose pathway is located in the mitochondria, while theterminal enzyme in the myo-inositol and L-gulose routes (GulLO) is knownto reside in the endoplasmic reticulum. All other enzymes are supposedto be cytoplasmic. However, the localization of the third enzyme (GNL)is unknown. In 2004 gene sequences of well characterized GNLs from ratand bacteria were aligned and compared to the Arabidopsis genome (A.Lorence, personal communication). This resulted in the identification of18 putative GNL candidate genes. FIG. 5 shows the Putativeglucuronolactonases (GNLs) in Arabidopsis. The T-DNA knockouts werescreened looking for low AsA lines to identify true GNLs in Arabidopsis.The foliar AsA content in knockout lines corresponding to the GNLArabidopsis genes were measured to identify low AsA mutants.Bioinformatics analysis of the genes found that one of the SALK lineswith low AsA encodes a protein that possesses a chloroplastic signalpeptide. In addition, microarray data available at Genevestigator(Zimmermann et al., 2004) showed that there is a suppression of theexpression of this gene (At1g56500) when plants are exposed to darkconditions. To understand the role of AtGNL in plant physiology, twoknockout lines with a T-DNA inserted into the At1g56500 gene wereobtained from the Arabidopsis Biological Resource Center (ABRC). FIG. 6shows the Schematic of the insertion site of the T-DNA in the At1g565400gene in SALK lines 026172 and 011623 (red squares). Exons are shown asblue boxes. Source: TAIR database.

The At1g56500 cDNA was amplified and sub-cloned into the pBIB-Kan vectorunder the control of the cauliflower mosaic virus 35S promoter and thetobacco etch virus (TEV) enhancer. A 6×-HIS tag was added at the 5′ endof the cDNA to facilitate protein detection by Western blot andpurification by nickel affinity chromatography.

To confirm that indeed this gene encodes a protein residing in thechloroplast, the Nicotiana benthamiana was infiltrated with an AtGNLconstruct using an optimized Agrobacterium-mediated transienttransformation method (Medrano et al., 2009). Chloroplasts were isolatedfrom leaves using a chloroplast isolation kit (CP-ISO Sigma). A Westernblot developed with an anti-HIS antibody, confirmed that AtGNL is indeedin the chloroplast as illustrated in FIG. 7. FIG. 7 shows the AtGNLprotein resides in the chloroplast. Chloroplasts were isolated fromleaves of Nicotiana benthamiana plants infiltrated with the AtGNLconstruct using a chloroplast isolation kit (CP-ISO, Sigma). Westernblot was done using an anti-HIS antibody. M: molecular weight marker,lane 1 empty vector fraction, lane 2 non chloroplastic fraction, lane 3chloroplastic fraction, lane 4 chloroplast fraction after purificationby nickel affinity chromatography.

This work focuses on characterizing a functional At1g56500 (AtGNL). Inthis work three aims are proposed: Aim 1: Characterize the AtGNL(At1g56500) functional enzyme, Aim 2: Establish the role of the AtGNLunder low, normal, and high light conditions, and Aim 3: Characterizethe phenotype and photosynthetic efficiency of Arabidopsis lines withlow, normal, and high AtGNL expression.

Vitamin C (L-ascorbic acid, AsA) is the most abundant water-solubleantioxidant in plants. Ascorbate scavenges free radicals, is an enzymecofactor, and a donor/acceptor of electrons in the chloroplast.Ascorbate protects tissues against damage caused by reactive oxygenspecies (ROS) produced through normal metabolism or generated fromstress. The inositol route to AsA involves four enzymes: myo-inositoloxygenase, glucuronate reductase, gluconolactonase (GNL), andL-gulono-1,4-lactone oxidase (GulLO). The third enzyme, GNL, has beencharacterized in rat and bacteria but not in plants. Eighteen putativeGNLs were identified in Arabidopsis, one of which, AtGNL, is interestingbecause it possesses a chloroplastic signal peptide. Chloroplasts canaccumulate up to 50 mM AsA but until now no chloroplastic AsAbiosynthetic genes have been described. This study includes thecharacterization of the first plant GNL enzyme in vitro and in planta.Knockouts on this gene had lower foliar AsA and stunted growth comparedto controls. The functional gene restored the phenotype of theknockouts, and those plants had higher AsA content, and enhancedphotosynthetic capacity. These results highlight the importance of AtGNLin AsA formation and in maintaining a healthy redox balance in theleaves particularly under low light stress. AtGNL is the first AsAbiosynthetic enzyme that resides in chloroplasts.

Accordingly, it is an object of the present invention to characterizethe AtGNL (At1g56500) functional enzyme.

It is another object of the present invention to establish the role ofthe AtGNL under low, normal, and high light conditions.

It is another object of the present invention to characterize thephenotype and photosynthetic efficiency of Arabidopsis lines with low,normal, and high AtGNL expression.

It is another object of the present invention to increase photosyntheticefficiency.

It is another object of the present invention to increase AtGNLexpression.

It is another object of the present invention to provide a method for apurification protocol for AtGNL recombinant protein.

It is another object of the present invention to increase the levels ofAsA within a plant.

It is another object of the present invention to increase the biomass ofthe plants.

It is another object of the present invention to delay aging of theplants.

It is another object of the present invention to increase production ofATP.

These and other objects and advantages of the present invention, alongwith features of novelty appurtenant thereto, will appear or becomeapparent in the course of the following descriptive sections and theattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following drawings, which form a part of the specification andwhich are to be construed in conjunction therewith, and in which likereference numerals have been employed throughout wherever possible toindicate like parts in the various views:

FIG. 1 is a chemical structure view of one embodiment of the presentinvention;

FIG. 2 is a metabolic network view thereof;

FIG. 3 is a flowchart view thereof;

FIG. 4 is a metabolic network view thereof;

FIG. 5 is a table view thereof;

FIG. 6 is a schematic view thereof;

FIG. 7 is a blot view thereof;

FIG. 8 is a construct view thereof;

FIG. 9 is a table view thereof;

FIG. 10 is a blot view thereof;

FIG. 11 is a blot view thereof;

FIG. 12 is an analytical view thereof;

FIG. 13 is a table view thereof;

FIG. 14 is a graph view thereof;

FIG. 15 is a graph view thereof;

FIG. 16 is a graph view thereof;

FIG. 17 is a table view thereof;

FIG. 18 is a graph view thereof;

FIG. 19 is a photographic and chart view thereof;

FIG. 20 is a photographic view thereof;

FIG. 21 is a chart view thereof;

FIG. 22 is a table view thereof;

FIG. 23 is a chart view thereof;

FIG. 24 is a chart view thereof;

FIG. 25 is a metabolic network view thereof;

FIG. 26 is a chart view thereof;

FIG. 27 is a table view thereof;

FIG. 28 is a chart view thereof;

FIG. 29 is a table view thereof;

FIG. 30 is a chart view thereof;

FIG. 31 is a table view thereof;

FIG. 32 is a photographic view thereof;

FIG. 33 is a phylogenetic view thereof;

FIGS. 34A and 34B are table views thereof;

FIG. 35 is a chart view of one embodiment of the present invention;

FIGS. 36A, 36B, and 36C are a genetic sequencing view of one embodimentof the present invention (SEQ ID NO: 1); and

FIG. 37 is a genetic sequencing view of one embodiment of the presentinvention (SEQ ID NO: 2).

DETAILED DESCRIPTION

Arabidopsis thaliana ecotype Columbia wild type seeds (Col-0, stock#CS60000), SALK_026172, and SALK_011623 were obtained from theArabidopsis Biological Resource Center (ABRC, Columbus, Ohio). Seedswere sterilized with 70% (v/v) ethanol for 10 min followed by 50% (v/v)sodium hypochlorite containing 0.05% (v/v) Tween-20 for 15 min. Next,seeds were washed 6 times with sterile water. Finally, seeds weretransferred to a petri dish containing medium which consisted ofMurashige-Skoog (MS) salts (Murashige and Skoog, 1962), MS vitamins, and3% (w/v) sucrose, at pH 5.6. The medium was supplemented with 0.04%(w/v) MgSO₄.7H₂O. The seeds were vernalized for 3 days at 4° C. Plateswere transferred to a growth chamber and incubated at 23° C., 65%humidity, 16:8 h photoperiod and 200 μmol/m²/s light intensity. Afterestablishment, seedlings were transferred to PM-15-13 AIS MIXArabidopsis soil (Lehle-Seeds, Round Rock, Tex.) in 2 inch pots. Potswere covered with a dome for one week and after that plants were grownuntil they reached maturity.

Nicotiana benthamiana seeds were obtained from The Department of PlantPathology, Physiology and Weed Science at Virginia Polytechnic Instituteand State University (Blacksburg, Va.). Seeds were sown in 4.5 inch potscontaining Pro-mix BX soil (Premier Horticulture Ltd, Canada) withfertilizer Osmocote 14-14-14 (Scotts, Canada). Vermiculite was overlaidon top of the seeds. The pots were covered with a dome for one week.Plants were grown in an environmental control chamber with the followingconditions: 25° C. (day)/21° C. (night) temperature, 65% relativehumidity, 16:8 h photoperiod, and 150 μmol/m²/s light intensity.

2.2.2. Constructs of Interest

Two gene constructs were made for this project. The first construct isone where the cDNA encoding a putative AtGNL was placed under thecontrol of the 35S promoter and the tobacco etch virus (TEV) enhancer(AtGNL-6×HIS:pBIB-Kan), (Becker, 1990). In this construct a histidinetag was added to the C-terminus of the protein of interest to allowdetection using antibodies and to facilitate purification. The secondconstruct is one where the putative promoter region of the AtGNL (a 1000bp fragment preceding the ATG) was cloned and fused to the GUS reportergene to better understand the spatial and temporal expression of thisgene (pAtGNL:pCAMBIA1305.1). FIG. 8 shows the Constructs of interest.(A) The At1g56500-HIS:pBIB-Kan construct containing At1g56500 (AtGNL)with a six histidine (6×-HIS) tag and adjacent neomycinphosphotransferase II (nptII) selectable marker. (B) ThepAt1g56500:pCAMBIA1305.1 construct containing the AtGNL promoter withthe GUS-PLUS reporter gene and the hygromycin phosphotransferase (hph)selectable marker. NOS-P: promoter of nopaline synthase gene, 35S-T:terminator of the 35S cauliflower mosaic virus gene; TEV: tobacco etchvirus translational enhancer; LB and RB: left and right T-DNA borders,respectively.

2.2.3. Development of Transgenic Lines

To study the expression of AtGNL in Arabidopsis thaliana, stabletransgenic plants were developed by the floral dip method (Clough andBent, 1998). Wild type CS60000 and knockouts: SALK_026172 andSALK_011623, were transformed with the Agrobacterium tumefaciens GV3101strain carrying the construct of interest. The T0 seeds were planted onMS medium plus kanamycin. The antibiotic resistant seedlings weretransferred to soil and grown to maturity under the above statedconditions. The presence of the transgene of interest was establishedvia PCR using gene specific primers, and genomic cDNA as a template. Todevelop homozygous versions of the knockout lines, over-expresser (wildtype plus AtGNL), and restored lines (knockouts plus AtGNL) T1 plantsthat were high AsA expressers were identified. The seeds of those plantswere sterilized with 70% (v/v) ethanol for 10 min followed by 50% (v/v)sodium hypochlorite containing 0.05% (v/v) Tween-20 for 15 min. Next,seeds were washed 6 times with sterile water. Finally, seeds weretransferred to petri dishes containing Murashige and Skoog (MS) mediumwhich consists of salts, MS vitamins, and 3% (w/v) sucrose at pH 5.6.The medium was supplemented with 0.04% (w/v) MgSO₄.7H₂O, and 50 mg/Lkanamycin. Plated seeds were vernalized for 3 days at 4° C. Afterestablishment, seedlings were transferred to soil and grown under theabove stated conditions until they reached maturity. This process wasrepeated until plants with a 100% germination score in the presence ofantibiotic selection were developed.

2.2.4. Ascorbate Measurements

In Arabidopsis, in planta AsA concentration changes throughout the dayas well as during development (Tamaoki et al., 2003; Zhang et al.,2009). Fifty mg of leaf tissue were collected between 9:00 am and 11:00am. Tissue was frozen immediately in liquid nitrogen and stored at −80°C. until analyzed. Reduced, oxidized, and total AsA were measured usinga 96-well plate format as described by Haroldsen et al., (2011).Briefly, frozen tissue was pulverized in 6% (w/v) meta-phosphoric acid,and centrifuged at 13,000 rpm for 15 min. Reduced AsA was determined bymeasuring the decrease in absorbance at 265 nm after addition of 0.5unit of ascorbate oxidase to 300 μL of the reaction medium containingthe plant extract and 100 mM phosphate buffer at pH 6.9. Oxidizedascorbate was measured in a 300 μL reaction mixture with 10 μL of 40 mMdithiothreitol (DTT) after incubation in the dark for 20 min at roomtemperature. The reaction was followed by measuring absorption at 265nm. Calculations were made based on a standard curve made with pureL-ascorbic acid run in parallel. Ten biological replicates were measuredin analytical triplicate and reported as μmol per gram fresh weight(μmol/g FW).

2.2.5. Transient Expression of AtGNL in Nicotiana benthamiana

In order to test its function the putative GNL recombinant protein wasproduced in Nicotiana benthamiana. This plant is a widely used platformfor the production of active proteins, including antibodies, enzymes andother proteins that require post-translational modifications (Klimyuk etal., 2012; Leuzinger et al., 2013). To find the optimal expression time,five week old N. benthamiana plants were vacuum infiltrated with theAt1g56500-6×HIS.pBIB-kan construct as described by Medrano et al., 2009.Leaf tissue was harvested at 24, 48, 72, and 96 h post infiltration forfurther analysis. The optimum time for tissue collection based onWestern blot data, was found to be at 48 h post infiltration (data notshown). In subsequent experiments all leaves were collected at 48 hpost-infiltration, frozen immediately in liquid nitrogen and stored at−80° C. until further processing. Plants infiltrated with the emptypBIB-kan binary vector (EV) were used as controls for these experiments.

In order to confirm the expression of the AtGNL in N. benthamiana,Western blot experiments were carried out. Crude extracts were made bygrinding frozen tissue in the presence of two volumes of SDS buffercontaining 150 mM Tris-HCl pH 6.8, 5 mM EDTA pH 8.0, 30% (v/v) glycerol,6% (w/v) SDS. The homogenate extract was then centrifuged at 13,000×gfor 15 min, and the supernatant was recovered. Proteins were separatedvia SDS-PAGE. Six L of plant extract were mixed with 2.5 μL of SDSloading buffer (4×) and 1 μL of DTT, incubated for 10 min at 70° C. andseparated by SDS-PAGE on 10% precast mini-gels (Expedeon, San Diego,Calif.) with a Tris-MOPS buffer. Subsequently, separated proteins wereelectro blotted onto a nitrocellulose membrane, using transblottingbuffer containing: 25 mM Tris base, 192 mM glycine, and 20% methanol.Recombinant AtGNL-6×HIS was detected using an anti-HIS (C-term)/APantibody at a 1:2,000 v/v dilution (Invitrogen, Carlsbad, Calif.) andCDP-start, a chemiluminescent substrate for alkaline phosphatasedetection (Roche Diagnostics, Indianapolis, Ind.).

2.2.6. Recombinant AtGNL Purification

Recombinant AtGNL protein was purified from N. benthamiana leaves. Fivegrams of leaf tissue were pulverized in liquid nitrogen and proteinswere extracted with 10 mL of buffer A (75 mM sodium phosphate dibasic,25 mM sodium phosphate monobasic, 150 mM NaCl, 10 mM sodiummetabisulfite, and 0.6% (v/v) protease inhibitor cocktail, pH 7.4). Theextract was then centrifuged at 13,000×g for 15 min. The supernatantobtained after centrifugation was loaded onto a nickel affinity column(HIS60 Ni Superflow) and incubated for 1 h at 4° C. Then, the column waswashed with 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 40 mM imidazolebuffer and the bound proteins were eluted with 250 mM of imidazole. Theeluate from the nickel column was concentrated using an AMICON® 30Kultra centrifugal filter (Millipore, Billerica, Mass.). Total solubleprotein concentration was estimated by the Bradford method (Bradford,1976) using Coomassie blue G-250 dye (Thermo Scientific) and bovineserum albumin (Pierce, Rockford, Ill.) as a standard. Protein fractionsfrom the purification procedure were separated by SDS-PAGE and the AtGNLwas detected by Western blot and silver staining using Pierce® SilverStain Kit (Thermo Scientific).

2.2.7. Recombinant AtGNL Enzyme Assay

The lactonase activity was assayed in vitro based on the decrease inabsorbance (405 nm) of the p-nitrophenol pH indicator that resulted fromthe enzymatic opening of the lactone ring when D-glucono-δ-lactone wasused as substrate in the presence of the AtGNL as previously described(Ishikawa et al., 2008). Enzyme preparations were made fresh forindividual experiments at room temperature.

In order to establish the optimal enzyme activity for AtGNL, severalconditions were tested. The optimum concentration for enzyme activitywas 30 μg per reaction. One mL of the reaction typically contained: 10mM PIPES pH 6.5, 5 mM D-glucono-δ-lactone, 75 μM MnCl₂, 2.5 mMp-nitrophenol, and an aliquot of the purified enzyme. An equal amount ofboiled enzyme was used as control for these experiments.

In order to examine the specificity of the AtGNL enzyme forD-glucono-δ-lactone, multiple substrates were tested in the lactonaseassay. The substrates used in this experiment were: D-glucono-δ-lactone(D-GuIL), L-galactono-γ-lactone (L-GaIL), L-galactonic acid (L-GalA),L-gulono-γ-lactone (L-GulL), and L-gulonic acid (L-GulA). The L-GaIA andL-GuIA were prepared by the hydrolysis of L-GaIL and L-GuIL,respectively. For hydrolysis 20 mL of 0.3 M NaOH were added to 100 μL of10 mM L-GaIL or L-GuIL, the mixture was vigorously agitated by vortexingfor 20 s, and 20 μL of 0.3 M HCl were added to neutralize the solution(Ishikawa et al., 2008). For enzyme kinetic experiments, individualreactions were monitored for 15 min at different substrateconcentrations. Analysis was done using the GraphPadPrism 6.2 software.

2.2.8. High Throughput Phenotyping

To characterize the phenotype of the over-expresser (L60, L61, L62),knockout lines (SALK_026172 and SALK_011623), and restored lines (L89,L90, L100, L128, L129, L130), under low, normal, and high lightconditions a high throughput phenotyping platform (Scanalyzer HTSinstrument, Lemnatec, Germany) and the LemnaControl software were used.This instrument is equipped with a robotic arm that holds visible (VIS,a.k.a. RGB), fluorescence (FLUO), and near infrared (NIR)high-resolution cameras. This system empowers unbiased, non-invasive,automated, and effective characterization of plant phenotypes. Thecameras in the system are as follows: VIS camera, piA2400-17gc CCD(Basler, Ahrensburg, Germany) with resolution of 2454×2056 pixels; FLUOcamera, scA1600-14gc CCD (Basler, Ahrensburg, Germany) with resolution1624×1234 pixels; and NIR camera, Goldeye GIGE P-008 SWIR (Allied VisionTechnologies, Stadtroda, Germany) with resolution 320×256 pixels andwith spectral sensitivity between 900 and 1700 nm.

In the greenhouse plants were grown in PM-15-13 AIS MIX soil(Lehle-Seeds, Round Rock, Tex.) in Quickpot 15 trays in a greenhouseduring Mar. 12-30, 2015 in Jonesboro, Ark., USA (latitude 29.4889 andlongitude −98.3987). Growth conditions were as follows: 22° C.-26° C.temperature, 16:8 h photoperiod, 55% humidity and three different lightconditions: low (35-110 μmol/m²/s), medium (110-350 μmol/m²/s) and highlight (350-700 μmol/m²/s). Light intensity was recorded four times perday (9:00 am, 12:00 pm, 3:00 pm, and 6:00 pm) to cover the entiresunlight period.

Images of AtGNL lines were captured every two days between 16 days and26 days after germination, to cover the full vegetative growth. Images(5670 images=7 lines×15 biological replicates×3 light treatment×6 timespoints×3 cameras) were analyzed using the LemnaGrid Software. Theanalysis of the RGB images was done as previously described by Arvidssonet al., (2011). Multiple phenotypic parameters were calculated for eachplant including: projected leaf area (cm²), convex hull area (cm²),caliper length (a.k.a. rosette diameter, mm) and compactness (measure ofthe bushiness of the plant). From the RGB images the relative area ofthe plants displaying normal green color versus the area with detectableyellow color (chlorosis) were calculated. The analysis of the NIR imageswas similar to the color classification of VIS camera, using theacquired gray-scale images, where high water content corresponds todarker tones while low water corresponds to lighter gray tones. Thesoftware used this information to calculate the relative area with low,medium, and high water content. The fluorescence camera acquiresred-scale images and in this case the red tones were divided into fourequidistant bins, and the software calculated the relative area withzero, low, medium, and high fluorescence. Quantitative data obtainedfrom the images were analyzed.

2.2.9. Photosynthetic Efficiency

In order to determine photosynthetic efficiency of photosystem II(Φ/II), linear electron flow (LEF), and non-photochemical quenching(NPQt) of the knockout lines (SALK_026172 and SALK_011623),over-expresser (L61), restored lines (L100 and L129), empty vector, andwild type controls growing under low and normal light conditions wereanalyzed using a MultispeQ. This is a hand held fluorometer developed bythe Kramer Laboratory at Michigan State University. Ten biologicalreplicates were chosen randomly at the same time of day formeasurements. Data were visualized in an Android tablet (Samsung GalaxyTab 4) and analyzed in the PhotosynQ website (www.photosynq.org).

2.2.10. PromoterAtGNL:GUS Expression in Arabidopsis thaliana

To study the expression of At1g56500 in different plant tissues,Arabidopsis thaliana var. Columbia was transformed by the floral dipmethod (Clough and Bent, 1998) with Agrobacterium tumefaciens GV3101carrying the construct of interest (pAtGNL:pCAMBIA1305.1). A differentset of plants was also transformed with bacteria carrying the emptyvector control (pCAMBIA1305.1). T0 seeds were selected with hygromycinand the antibiotic resistant seedlings were transferred to soil andgrown to maturity under the above mentioned conditions. The presence ofthe transgene of interest was established via PCR using gene specificprimers, and genomic cDNA as a template. Seeds of the PCR positiveplants were sterilized and transferred to a petri dish containing MSmedia with 20 mg/L hygromycin. Plated seeds were vernalized for 3 daysat 4° C. and then transferred to an environmentally controlled chamber.Hygromycin resistant seedlings were transferred to soil and grown untilmaturity.

Explants (seedlings, leaves, flowers, and fruits) were cut from plants4, 8, 12, and 30 days after germination. Next, the explants wereincubated in fresh and cold phosphate buffer pH 7.0 with 4% formaldehydeat room temperature for 30 min. The explants were washed several timeswith cold phosphate buffer for 1 h, then vacuum infiltrated with X-Glucsubstrate solution containing: 1 mg 5-bromo-4-chloro-3-indolylβ-D-glucuronide in 100 μL of methanol, 1 mL 2× phosphate buffer, 20 μL0.1 M potassium ferrocyanide, 20 μL 0.1 M potassium ferricyanide, 10 μL10% (w/v) solution of Triton X-100, and 850 μL of water. Tissues wereincubated in darkness at room temperature overnight until a distinctblue staining appeared. Finally, explants were incubated in 70% ethanoluntil the chlorophyll was removed. Photographs were taken with AxioCamMRc camera connected to a Stemi 2000-C stereo microscope (Zeiss).

2.2.11. Phylogeny

In order to identify a functional GNL in Arabidopsis thaliana (AtGNL),known GNLs, and putative GNLs from other organisms were compared usingthe TAIR database (www.arabidopsis.org). The AtGNL was obtained fromTAIR database based on highest protein homolog. That sequence was thenconverted to FASTA format using the EMBL-EBI(www.ebi.ac.uk/tools/stc/readseq/). The MEGA6 software enabled readingand comparing the AtGNL with known and putative GNL sequences (Tamura etal., 2013).

2.2.12. Statistical Analysis

Data was analyzed by SAS software 9.4 (SAS Institute, 2016). Analysis ofvariance was carried out by ANOVA procedure. Least squares means(LS-means) were calculated to evaluate AsA content per line, at α=0.05.

2.3. Results and Discussion

2.3.1. Purification and Characterization of Recombinant At1g56500

To demonstrate the GNL activity of Arabidopsis thaliana gluconolactonasein vitro, an N. benthamiana-based transient expression system was used.Plants were vacuum infiltrated with the Agrobacterium tumefaciensLBA4404 strain carrying the At1g56500-6×HIS construct. The proteinaccumulation is highest at 48 h post infiltration (data not shown).

In order to establish a protein purification protocol for AtGNL, severalextraction buffers were tested to identify those that allow recovery ofthe highest amount of protein. FIG. 9 shows the list of buffers testedfor protein purification.

Protein fractions from the various purification procedures wereseparated by SDS-PAGE and AtGNL was detected by Western blot. FIG. 10shows the AtGNL protein extracted with different buffers. Western blotof total protein extracted from N. benthamiana leaves with differentbuffers as described in FIG. 7. M: marker, lane 1: crude extract withbuffer-1, lane 2: crude extract with buffer-2, lane 3: crude extractwith buffer-3, lane 4: crude extract with buffer-4, lane 5: crudeextract with buffer-5, lane 6: sample extracted in buffer-5 andresuspended in buffer-4, lane 7: crude extract in buffer-5, lane 8:sample extracted in buffer-5 and resuspended in buffer-3, lane 9: sampleextracted in buffer-5 and resuspended in buffer-6, lane 10: crudeextract in buffer-6.

The optimal buffer to recover more recombinant protein was buffer 6.

In order to establish the optimal concentration for washing and elutionbuffer, several imidazole concentrations were tested. FIG. 11 shows theAtGNL protein eluted with different imidazole concentrations. Westernblot of AtGNL protein with different concentration of imidazole. M:marker, B6: crude extract in buffer 6, FT flow through, W: wash buffer.As illustrated in FIG. 11, 40 mM and 250 mM were best for washing andeluting conditions, respectively.

FIG. 12 shows the Purification of the AtGNL:pBIB-kan-6×HIS expressed inN. benthamiana leaves. M: marker, lane 1: crude extract, lane 2: flowthrough, lane 3: wash, lane 4: enzyme, lane 5: concentrated enzyme. FIG.12 illustrates the result of the purification of AtGNL from N.benthamiana tissue using nickel affinity chromatography. Western blotresults showed the presence of AtGNL in the crude extract and flowthrough or wash indicating protein had a partial binding to the cationcolumn. The silver-stained gel indicates that the protein preparationcontained mostly the protein of interest with a few minor contaminants.

2.3.2. Enzyme Activity of Recombinant At1g56500

Once an effective purification procedure was developed, the next stepwas to standardize the assay to test the AtGNL activity.Gluconolactonase (GNL, EC 3.1.1.17) catalyzes the hydrolysis ofD-glucono-σ-lactone (D-GulL) to D-gluconic acid (Ogawa et al., 2002).The lactonase activity was assayed in vitro based on the decrease inabsorbance (405 nm) of the p-nitrophenol pH indicator that resulted fromthe enzymatic opening of the lactone ring when D-glucono-δ-lactone(D-GuIL) was used as substrate in the presence of the AtGNL aspreviously described (Hucho and Wallenfels, 1972). The enzymaticactivity was assayed at 25° C. with 10 mM PIPES pH 6.5, 5 mM D-GuIL, 75μM MnCl₂, 2.5 mM p-nitrophenol, and 30 μg of the purified enzyme (AtGNL)in 1 mL of reaction (Ishikawa et al., 2008). With the exception ofD-GuTL, the recombinant AtGNL did not exhibit activity with any of thesubstrates tested. FIG. 13 shows the Substrate preference of AtGNL.

All enzymes work with a range of temperatures specific to the organismfrom which they are extracted. The effect of temperature on the AtGNLactivity was also determined. The activity of the AtGNL enzyme washighest at temperatures between 25° C. and 35° C. The activitydrastically decreased when the temperature was increased to 40° C. FIG.14 shows the Effects of temperature and pH on the activity of the AtGNLenzyme. (A) pH effect on GNL activity. (B) Temperature effect on GNLactivity. Measurements were made in duplicate. Values are means±SD.Ogawa et al., (2002) reported that the GNL enzyme from A. niger hadhigher activity at 30° C., while the activity of the GNL from P.aeruginosa is optimal at 24° C. (Tarighi et al., 2008), which aresimilar to the AtGNL.

Kondo et al., (2006), reported that the activity of the rat GNL washighest at pH 6.4, while Tarighi et al., (2008) demonstrated that theoptimal activity of the P. aeruginosa GNL was at pH 7.2. In contrast, inthis study the A. thaliana GNL enzyme had a higher activity at pH 6.0,and the activity decreased by 4-fold when the pH was increased to 6.3(FIG. 14). Lower pH values were not tested because PIPES buffer cannotdissolve at pHs lower than 6.0.

To assess if the AtGNL activity had a preference for a particulardivalent ion, various cofactors were tested. Ishikawa et al., (2008)reported that the GNL enzyme from E. gracilis had a higher activityusing ZnCl₂ as a cofactor and that this activity decreased around 4-foldwhen changed to MnCl₂. In these experiments, no significant differencein GNL activity among the tested cofactors was observed (FIG. 15).Increasing the substrate concentration increased the rate of reaction orenzyme activity. In order to identify the optimal concentration of theD-glucono-δ-lactone, multiple substrate concentrations were tested. FIG.15 shows the Effects of cofactor and substrate on the activity of theAtGNL enzyme. (A) Cofactors effect on GNL activity. (B) D-GuIL substrateconcentration effect on GNL activity. Measurements were made induplicate. Values are means±SD. The 3 mM of D-glucono-δ-lactone was themost effective substrate concentration for this assay.

Enzyme kinetic analysis was performed with D-GuIL at a concentration of1 mM to 50 mM of substrate. The enzyme activity with 5 mM of D-GuIL atpH 6.0 was 10.54 μmol min⁻¹ mg⁻¹ of protein, V_(max)=1.161×10⁻⁶ (38.7μmol min⁻¹ mg⁻¹ of protein) and K_(m)=2.989. FIG. 16 shows the Enzymekinetics of the recombinant At1g56500 enzyme. (A) Michaelis-Menten. (B)Double reciprocal Lineweaver-Burke. Measurements were made in duplicate.Values are means±SD. FIG. 17 summarizes the comparison between thekinetic parameters of the AtGNL with the one of known GNLs. Based onthese results the G. oxidans GNL is the most similar to the ArabidopsisGNL.

2.3.3. Characterization of the Phenotype of Gluconolactonase Lines witha Scanalyzer HTS Platform

Seeds expressing the AtGNL-6×HIS:pBIB-kan (AtGNL) and empty pBIB-Kan(control) were screened in the Lorence Laboratory (unpublished). Onehundred and thirty primary transformants that were PCR positive werescreened to identify high AsA expressers. After four rounds ofscreening, three lines per group were selected for further analysis:over-expressers (WT+AtGNL), restored 1 (SALK_026172+AtGNL), and restored2 (SALK_011623+AtGNL).

Homozygous lines (T5), plants with 100% germination in the presence ofantibiotic selection were developed for over-expresser (L60, L61, L62),restored-1 lines (L89, L90, L100), and restored-2 lines (L128, L129,L130). FIG. 18 shows the total foliar AsA level of AtGNL lines undernormal light conditions. (A) Over-expressers and wild type (WT). (B)Restored lines and knockout control (S_026172). (C) Restored lines andknockout control (S_011623). Asterisks indicate significant differencesbetween controls and high AsA lines as determine by Turkey multiplecomparisons test, α=1, ****=P<0.0001, ***=P<0.0002, **=P<0.0017,*=P<0.0155. Values are means±SD, n=15.

The phenotype of these homozygous lines was analyzed using a ScanalyzerHTS instrument under normal conditions as described in materials andmethods. Plant images were captured every two days from 16 to 26 daysafter germination. Representative images of homozygous AtGNL lines andtheir respective controls are shown in FIG. 19. From these images theprojected leaf area as an indicator of plant growth was measured. FIG.19 shows the Phenotype of AtGNL lines grown under normal conditions. (A)Representative images of AtGNL lines acquired with the visible camera(aka RGB). (B) Growth curves of AtGNL lines compared with theirrespective controls. Values are means±SE, n=15. There is a strongcorrelation between higher biomass and projected leaf area. Restored-1and restored-2 lines had more biomass compared to their controlsSALK_026172, SALK_011623, respectively, and those restored linespresented higher projected leaf area compared with their controls.

Based on these results, foliar AsA level, and the phenotype analysis,further studies were done only with the lines that had the highestfoliar AsA content and fastest growth and higher biomass and projectedleaf area.

Over-expresser L61 (OE) and the empty vector control (EV); restored-1L100 (R-1) and its control SALK_026172 (KO-1), restored-2 L129 (R-2),and its control SALK_011623 (KO-2), and wild type (WT) control wereselected for further analysis. First, the effect of low and high lightconditions on the selected plants was assessed. Routinely, plants aregrown in environmental control chambers. A first attempt to study lighteffects was accomplished by growing plants in multiple chambers: (315=7lines×15 biological replicates×3 light treatments). However, it was verydifficult to achieve uniform conditions, with the only variable beingthe light intensity. To solve this problem, the experiment was conductedin the greenhouse. Two different density meshes were used to diffuse thelight to the plants. FIG. 20 shows the experimental set up for studyingthe effect of light on the phenotype of AtGNL lines. Light intensity wasmeasured four times per day (9:00 am, 12:00 pm. 3:00 pm, and 6:00 pm) tocover the sunlight period. The light intensity for these three treatmentwas defined as: low light (35-110 μmol/m²/s), normal light (110-350μmol/m²/s), and high light (350-700 μmol/m²/s).

Normal outdoor light on a sunny summer day is around 1000-2000 μmol/m²/s(Mishra et al., 2012) However, because A. thaliana is a springunderstory plant, anything above 350 μmol/m²/s is considered high light.

Fifty mg of leaf tissue were collected at developmental stage 6.3 asdefined by Boyes et al., (2001) between 9:00-11:00 am. Reduced,oxidized, and total AsA were measured via an enzyme-based method aspreviously described (Haroldsen et al., 2011). The results indicate thatthe over-expresser and the restored lines had a higher foliar AsA thantheir respective controls growing under similar conditions. FIG. 21shows the Total foliar AsA levels of AtGNL lines under low, normal andhigh light conditions. (A) Over-expressers and wild type (WT). (B)Restored lines and knockout control (SALK_026172). (C) Restored linesand knockout control (SALK_011623). S026172 had a lower significantdifference compared with wild type control at high light treatment. Eachline was compared to the control (WT), analyzed by t-tests (LDS) atα=0.05. Significant differences are indicated by ***. WT: wild type, EV:empty vector, OE: over-expresser, KO: knockout, R: restored. n=15. Thestatistical analysis of total foliar AsA content in the three differentlight treatments in AtGNL lines shown in FIG. 22 validates thisconclusion. Two-way ANOVA (α=0.05). The table in FIG. 22 indicates linesare significantly different and that there is a significant interactionbetween the lines and light treatments.

The projected leaf area results showed the same trend, whereover-expressers and restored lines were bigger than their controls, withKO-1 being the worst performer at all light conditions tested. FIG. 23shows the Projected leaf area of AtGNL lines grown under low, normal,high light conditions. (A) Low light. (B) Normal light. (C) High light.Values are means of 15 biological replicates. WT: wild type, EV: emptyvector, OE: over-expresser, KO: knockout, R: restored. n=15. There is astrong correlation between foliar AsA level and projected leaf area:over-expresser and restored lines had higher foliar AsA levels andhigher projected leaf area compared with their respective controls.

In planta chlorophyll, fluorescence measured with the fluorescencecamera can serve as an indicator of whether the plants are under stress.These plants were grown under the normal light regime. High fluorescencein plants is opposite of high photosynthetic efficiency (Lichtenthaler,1988). FIG. 24 shows the Chlorophyll fluorescence patterns of AtGNLlines. Relative in planta chlorophyll content measured with the FLUOcamera. Values are means of 15 biological replicates. WT: wild type, EV:empty vector, OE: over-expresser, KO: knockout, R: restored. n=15. Theknockout KO-1 line showed high fluorescence compared to the other linesunder high light conditions. This knockout line has a lower AsA level inthe leaves, lower biomass, and projected leaf area, and also higherfluorescence compared with rest of the lines. Overall these results showthat the AtGNL enzyme is essential to support normal AsA content inleaves and normal growth and development in Arabidopsis.

2.3.4. Photosynthetic Efficiency of AtGNL Lines Under Low and NormalLight Conditions

Genes involved in the AsA metabolic network have been identified thatwere down and up regulated in response to light. FIG. 25 shows theEffect of darkness on the expression of genes in the AsA metabolicnetwork. Microarray data deposited at Genevestigator was mined. Genesthat are down regulated in darkness are shown in red, while genes thatare up-regulated are shown in green. Yellow color indicates genesisoforms that are upregulated under dark conditions. TheD-mannose/L-galactose, L-gulose and D-glucuronate pathway are repressedunder low light conditions while the myo-inositol pathway keeps working.Suza and Lorence, unpublished. These results indicate that the L-gulose,D-mannose/L-galactose, and D-galacturonate pathways are down regulatedunder darkness, while the myo-inositol route is up regulated. If thetranscripts are down regulated, the enzymes are expected to be downregulated as well. Because photosynthetic efficiency is a measure oflight stress and redox potential, photosynthetic efficiency was analyzedfor AtGNL lines growing under low and normal light conditions. Thesephotosynthetic efficiency measurements were done with a MultispeQ, ahand-held device developed in the Kramer Laboratory (Michigan State).

Photosynthetic efficiency is the fraction of light (photons) that plantsobtain from the sun to convert into chemical energy duringphotosynthesis. Under normal light conditions there was no penalty inthe photosynthetic efficiency of plants lacking AtGNL expression. FIG.26 shows the Photosynthetic efficiency of AtGNL lines under low andnormal light conditions. Each line was compared to the control (WT),analyzed by t-tests (LDS) at α=0.05. Significant differences areindicated by ***. WT: wild type, EV: empty vector, OE: over-expresser,KO: knockout, R: restored. n=10. In contrast, the over-expressers andrestored lines displayed enhanced efficiency indicating a positiveimpact on photosynthesis due to higher AtGNL expression. When plantswere grown under low light, results were quite different. In this case alower photosynthetic efficiency was detected in EV and KO-1 compared tothe WT control. This indicates a penalty in photosynthetic efficiencydue to lack of AtGNL expression. FIG. 27 shows the Statistical analysisof photosynthetic efficiency of AtGNL lines grew at low and normal lightconditions. Two-way ANOVA (α=0.05). Photosynthetic efficiency asresponse to light conditions. The table indicates the treatment lighthas a significant effect pvalue=0.001. The lines also has a significanteffect pvalue<0.0001, and there is a significant interaction betweenlight and lines pvalue=0.1773.

In addition to photosynthetic efficiency two other parameters related tophotosynthesis were measured: linear electron flow (LEF) andnon-photochemical quenching (NPQt). The linear electron flow rate (LEF)has a direct correlation to photosynthetic efficiency. LEF facilitatesthe movement of H⁺ ions across the thylakoid membrane to create anelectrochemical gradient that is used by ATP-synthase to produce energy(ATP). FIG. 28 shows the Linear electron flow of AtGNL lines under lowand normal light conditions. Each line was compared to the control (WT),analyzed by t-tests (LDS) at α=0.05. Significant differences areindicated by ***. WT: wild type, EV: empty vector, OE: over-expresser,KO: knockout, R: restored. n=10. FIG. 28 shows that under low lightconditions both knockouts had lower LEF values than the controls, whileKO-1 was the line with the worst performance under normal lightconditions. FIG. 29 shows the Statistical analysis of linear electronflow of AtGNL lines grown at low and normal light conditions. Two-wayANOVA (α=0.05). LEF as response to light conditions. The table indicatesthe treatment light has a significant effect pvalue=0.0006. The linesalso has a significant effect pvalue<0.0001, and there is a significantinteraction between light and lines pvalue=0.0091. These data highlightsthe importance of the AtGNL enzyme for efficient ATP production in thechloroplasts.

Plants exhibit phenotypic plasticity and respond to differences inenvironmental conditions by acclimation. In a recent study, Arabidopsisplants grown under field conditions were compared with plants grownindoors. Indoor-grown plants had larger leaves, modified leaf shapes andlonger petioles and less NPQt, while field-grown plants had a highcapacity to perform state transitions (Mishra et al., 2012). Ifphotosynthesis is inefficient, excess light energy is dissipated as heatto avoid damaging the photosynthetic apparatus. When plants are underabiotic stress, such as low light intensity, the photosyntheticefficiency and the NPQt are opposite. The KO-1 line had high NPQt,indicating inefficient photosynthesis at both low and normal lightconditions. FIG. 30 shows the Non-photochemical quenching coefficient ofAtGNL lines under low and normal light conditions. Each line wascompared to the control (WT), analyzed by t-tests (LDS) at α=0.05.Significant differences are indicated by ***. WT: wild type, EV: emptyvector, OE: over-expresser, KO: knockout, R: restored. n=10. Statisticalanalysis indicates the KO-1 line had a highly significant differencecompared to the WT control. FIG. 31 shows the Statistical analysis ofnon-photochemical quenching of AtGNL lines grown at low and normal lightconditions. Two-way ANOVA (α=0.05). Non-photochemical quenching asresponse to light conditions. The table indicates the treatment lighthas a significant effect pvalue=0.001. The lines also has a significanteffect pvalue<0.0001, and there is a significant interaction betweenlight and lines pvalue=0.4475. Overall, the over-expressers, restoredlines, wild type, and empty vector lines had higher values ofphotosynthetic efficiency and LEF compared with KO lines under normaland low light conditions, while the NPQt values were opposite with theKO-1 having the highest value. These results show that AtGNL expressionis essential to maintain high photosynthetic efficiency, high electronflow to make ATP (high LEF) and less loss of energy in the form of heat(NPQt).

2.3.5. Temporal and Spatial Expression of AtGNL Using the GUS ReporterGene

To examine the expression of AtGNL within tissues, ten transgenic plantsexpressing GUS driven by the AtGNL promoter (pAt1g56500:pCAMBIA1305.1)and empty vector pCAMBIA1305.1 (control) were generated. In the emptyvector the GUS-PLUS gene is under the control of the 35S constitutivepromoter.

AtGNL, empty vector and wild type plants were treated with the X-Glucsubstrate. As illustrated in FIG. 32 GUS activity was evident in plantsexpressing the AtGNL promoter in all developmental stages fromcotyledons to roots, although much less staining was observed in4-day-old seedlings compared with the controls. The oldest seedlingsstained most intensely, especially at the leaf tips and margins. FIG. 32shows the Temporal and spatial expression of AtGNL using the GUS-PLUSreporter gene. The AtGNL is expressed in the whole plant and at alldevelopmental stages, indicating that the GNL enzyme is important in thephysiological development of the plant from beginning to maturity.

2.3.6. Phylogenetic Three of Putative Plant Gluconolactonases

A phylogenetic tree for At1g56500 (AtGNL) was generated. At1g56500(AtGNL) was compared with known GNLs and with putative GNLs for manyother organisms. After all protein sequences with significant sequencesimilarity to AtGNL were retrieved, the protein sequences were alignedusing the MEGA6 software (Tamura et al., 2013). Only the sequences thathad between 90 and 100% of identity with the AtGNL protein of interestwere included in this analysis. To develop an updated phylogenetic tree,a BLASTP search was done against the Arabidopsis protein database(www.arabidopsis.org) using the A. thaliana gluconolactonase “At1g56500”(AtGNL) protein sequence. This enzyme has been characterized in A. niger(Ogawa et al., 2002), E. gracillis (Ishikawa et al., 2008), P.aeruginosa (Tarighi et al., 2008), R. norvegicus (Kondo et al., 2006),Z. mobilis (Pedruzzi et al., 2007), and now also in Arabidopsis (thiswork). The BLASTP result revealed the presence of 37 candidates indifferent organisms with 90-100% identity to the AtGNL query. FIG. 33shows the Phylogenetic analysis of known and putative GNLs. Phylogeneticanalyses were conducted in MEGA6 (Tamura et al., 2013). Five branchescan be distinguished in this phylogenetic tree where the AtGNL groupswith proteins from plant species including plant crops of agriculturalimportance including Cucumis sativus (cucumber), Cucumis melo (melon),Citrus sinensis (orange), Vitis vinifera (grapes), Theobroma cacao(cacao), Glycine max (soybean), and Fragaria vesca (strawberry), treessuch as Populus trichocarpa (poplar) and Prunus mume (Chinese plum), andenergy crops such as Jatropha curcas. The sequence similarity betweenAt1g56500 and putative GNLs from other plants is remarkable. In a secondgroup we can see known GNLs from mammalian species including R.novergicus, H. sapiens, and putative GNLs from Ovis aries, Canisfamiliaris and others. In the third and fourth branches we can see knownand putative GNLs from bacteria and fungi. In a fifth group we findputative GNLs from A. thaliana. The analyses showed that At1g56500 hashigh similarity with the protein sequences listed in FIGS. 34A and 34B.FIGS. 34A and 34B show the List of known and putative GNLs included inthe phylogenetic analysis. Underlined species indicate the GNL enzymesthat have been characterized biochemically.

The constitutive expression of the gene of interest (GNL) leads tohigher seed yield in plants, such as Arabidopsis. Such higher seed yieldis shown in FIG. 35.

FIGS. 36A, 36B, and 36C show the GNL DNA sequence.

FIG. 37 shows the GNL amino acid sequence. In one embodiment, the GNLover-expressed in the plant may include at least 70% of the sequenceshown in FIG. 36 or FIG. 37.

2.4. Conclusions

The evidence presented in this document allows the followingconclusions:

We successfully developed a purification protocol for AtGNL recombinantprotein, and characterized this enzyme in detail including temperature,pH, cofactor requirement, and substrate concentration preferences, aswell as its kinetic parameters.

The AtGNL enzyme had highest activity at temperatures between 25° C. to35° C., while the bacteria P. aeruginosa has optimal temperatureactivity at 24° C. The optimum temperature of the AtGNL is consistentwith the preferred growth temperature of Arabidopsis.

The optimal pH for AtGNL enzymatic activity was 6.0, and this activitydecreased 4× when the pH was slightly increased (pH 6.3). In contrast,mammalian GNLs isolated from rats, mice, and humans have an optimalactivity at pH 6.4. Our result is consistent with the prevalent pH inthe chloroplasts (Alberts et al., 2002), the organelle where thisprotein resides.

The enzyme characterized in this work is very specific with the D-GuILsubstrate. In contrast the recombinant GNLs from E. gracilis and R.norvegicus are promiscuous as they displayed activity with additionalsubstrates (Kondo et al., 2006; Ishiwaka et al., 2008b).

GNL enzymes require a divalent cofactor for activity. The AtGNLdisplayed similar activity when incubated with MnCl₂, MgCl₂, or ZnCl₂.In contrast, other GNLs such as the E. gracilis isoform prefer ZnCl₂ andactivity dropped significantly with other cofactors (Ishikawa et al.,2008). The ability of AtGNL to work with MnCl₂ is consistent with anenzyme that is active in the chloroplasts, as Mn is abundant in thatorganelle (Alberts et al., 2002).

Based on optimum pH, optimum T, and kinetic parameters of the known GNLsthe one that is the most similar to the one here characterized is the G.oxydans GNL.

After demonstrating GNL enzyme activity in vitro, the present inventionprovides a method of controlling the role of this enzyme in AsAbiosynthesis in planta. As shown in FIG. 18 when constitutivelyexpressed in wild type, this enzyme leads to over-expressers with up to3-fold increase in foliar AsA content. Two T-DNA insertion knockouts inthis gene (SALK lines) had reduced AsA content compared to the WTcontrol. When the functional gene was inserted into the knockoutbackground this led to plants with restored AsA content. Overall thisdata indicates that AtGNL is functional in planta.

Previous results in the Lorence Laboratory obtained using manualphenotyping showed that plants with high AsA accumulate more biomass,delayed aging and are tolerant to abiotic stresses (Lisko et al., 2013).To determine if the AtGNL lines display differences in growth rate andbiomass accumulation, a powerful high throughput phenotyping instrumentwas used to characterize the phenotype of lines with normal (WT), low(knockouts) and high (over-expresser and restores lines) GNL expression.A clear penalty in the growth rate and biomass accumulation of theknockouts exists. The restored lines grew as well or better than thecontrols. This result indicates that AtGNL is key to the plant tosupport normal growth and development.

Based on the fact that AtGNL is a chloroplastic enzyme (FIG. 7), to gaininsights about the physiological role of this protein in supporting thefunction of this organelle, the ability of plants with low, normal, andhigh AtGNL expression to adapt to light stress conditions was analyzed.It is well established that there is higher ROS production during lowand high light exposure (Sharma et al., 2012). Arabidopsisover-expressers and restored lines where AtGNL expression is enhanceddisplay higher AsA content compared to controls.

Detailed characterization of the phenotype of the AtGNL lines undernormal, low and high light conditions showed that over-expressers andrestored lines grew better and accumulated more biomass than theirrespective controls (FIG. 23). The line with the poorest performance wasKO-1, the genotype with the lowest AsA content. Interestingly in plantachlorophyll fluorescence analysis show that KO-1 displayed highchlorophyll fluorescence, an indicator of stress. Further analysis willhave to be done to determine why KO-1 has a stronger phenotype comparedto KO-2.

Plant growth and yield depend on plants maintaining high photosyntheticefficiency. To determine if the stunted growth we measured in theknockouts is due to deficiencies in photosynthesis, measurements weredone with a hand-held fluorometer. Interestingly, results show thatover-expressers and restored lines displayed enhanced photosynthesiscompared to controls, while KO-1 and KO-2 have decreased LEF andtherefore decreased ability to make ATP. Overall KO-1 display thepoorest performance for all photosynthetic parameters here measuredincluding non-photochemical quenching.

In order to analyze the expression of the AtGNL in plants tissues wedeveloped transgenic A. thaliana lines expressing the GUS-PLUS reportergene under the control of the AtGNL promoter (pAtGNL). The results showthat AtGNL is expressed in all tissues examined: seedlings, leaves,stems, and siliques, except roots. This expression pattern suggests thatAtGNL is a constitutive enzyme.

AtGNL is an important enzyme to sustain sufficient AsA content and tomaintain plant growth and efficient photosynthesis. In order to gaininsights about the conservation of this enzyme in evolution, aphylogenetic tree of known and putative GNLs was developed (FIG. 33). Atleast 37 candidates with 90-100% sequence identity to the AtGNL at theamino acid level exist. This analysis indicates the presence of GNLs ina wide array of plants including crops of agricultural importance,mammals, bacteria, and fungi. Interestingly it appears to be a GNL inSellaginella moellendorffii, an ancient vascular plant that is widelyused as a model to study the evolution of plants as a whole (Banks etal., 2011).

From the foregoing, it will be seen that the present invention is onewell adapted to obtain all the ends and objects herein set forth,together with other advantages which are inherent to the structure.

It will be understood that certain features and sub-combinations are ofutility and may be employed without reference to other features andsub-combinations. This is contemplated by and is within the scope of theclaims.

As many possible embodiments may be made of the invention withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A method for increasing seed yield in a plantcomprising: expressing in a genetically engineered plant apolynucleotide encoding a gluconolactonase (GNL) polypeptide with atleast 90% sequence identity to SEQ ID NO:2 operably linked to aheterologous promoter to produce a plant with increased seed yieldrelative to a GNL wild-type plant and increased GNL expression relativeto a GNL wild-type plant wherein the GNL wild-type plant ischaracterized as having wild-type level expression of a wild-type GNLpolypeptide with at least 90% sequence identity to SEQ ID NO:2.
 2. Themethod of claim 1, wherein the promoter is a constitutive promoter. 3.The method of claim 2, wherein the promoter is cauliflower mosaic virus35S promoter.
 4. The method of claim 1, wherein the polynucleotide isoperably linked to an enhancer.
 5. The method of claim 4, wherein theenhancer is a tobacco etch virus enhancer.
 6. The method of claim 1,wherein the GNL polypeptide comprises SEQ ID NO:2.
 7. The method ofclaim 6, wherein the polynucleotide encoding the GNL polypeptidecomprises SEQ ID NO:1.
 8. The method of claim 1, wherein the plant istransformed with a construct comprising the polynucleotide encoding apolypeptide with at least 90% sequence identity to SEQ ID NO:2 operablylinked to a heterologous promoter.
 9. The method of claim 8, wherein theplant is transformed using Agrobacterium-mediated transformation.
 10. Agenetically engineered plant with increased seed yield and increasedgluconolactonase (GNL) expression comprising a construct, the constructcomprising a polynucleotide encoding a GNL polypeptide with at least 90%sequence identity to SEQ ID NO:2 operably linked to a heterologouspromoter, wherein seed yield and GNL expression of the geneticallyengineered plant is increased relative to a GNL wild-type plant of thesame species lacking the construct wherein the GNL wild-type plant ischaracterized as having wild-type level expression of a wild-type GNLpolypeptide with at least 90% sequence identity to SEQ ID NO:2.
 11. Thegenetically engineered plant of claim 10, wherein the constructadditionally comprises an enhancer.
 12. The genetically engineered plantof claim 11, wherein the enhancer is a tobacco etch virus enhancer. 13.The genetically engineered plant of claim 10, wherein the promoter is aconstitutive promoter.
 14. The genetically engineered plant of claim 13,wherein the promoter is cauliflower mosaic virus 35S promoter.
 15. Thegenetically engineered plant of claim 10, wherein the GNL polypeptidecomprises SEQ ID NO:2.
 16. The genetically engineered plant of claim 15,wherein the polynucleotide encoding the GNL polypeptide comprises SEQ IDNO:1.
 17. The genetically engineered plant of claim 10, wherein the GNLpolypeptide with at least 90% sequence identity to SEQ ID NO:2 istargeted to the chloroplast.
 18. A seed of the genetically engineeredplant of claim 10, wherein the seed comprises the construct.
 19. Amethod for producing the genetically engineered plant of claim 10comprising transforming a plant with the construct comprising apolynucleotide encoding a GNL polypeptide with at least 90% sequenceidentity to SEQ ID NO:2 operably linked to a heterologous promoter toproduce the genetically engineered plant.
 20. The method of claim 19,wherein the plant is transformed using Agrobacterium-mediatedtransformation.